Larvae were embedded in 1.5% agarose (low melting point) (Davis and Ramakrishnan, 2009 (link)). A series of z stack images with a 2 μm step size was generated through the infected HBV, using the galvo scanner (laser scanner) of the Nikon A1 confocal microscope with a 20x Plan Apo 0.75 NA objective. Bacterial burdens were determined by using the 3D surface-rendering feature of Imaris (Bitplane Scientific Software) (Yang et al., 2012 (link)).
Plan apo 0.75 na objective
Manufactured by Nikon
The Nikon 20x Plan Apo 0.75 NA objective is a high-performance microscope objective designed for advanced laboratory applications. It features a numerical aperture of 0.75 and a magnification of 20x, providing high-resolution imaging capabilities. The objective is part of Nikon's Plan Apo series, ensuring excellent optical flatness and color correction across the field of view.
Automatically generated - may contain errors
Lab products found in correlation
Orca flash 4.0 v2 cmos camera, by Hamamatsu Photonics (2 mentions)
24 well microplate, by Corning (2 mentions)
Plan apo 0.75 n objective, by Nikon (2 mentions)
A1 confocal microscope, by Nikon (1 mentions)
Water soluble cholesterol, by Merck Group (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
Lab tek 2 chambered coverglass, by Thermo Fisher Scientific (1 mentions)
P06g 1.5 20 f, by Thermo Fisher Scientific (1 mentions)
5 protocols using plan apo 0.75 na objective
1
Imaging and Quantifying Bacterial Infection
2
Cholesterol Modulation of M. marinum Infection
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A549 cells were seeded at 50,000 cells per 8-well Nunc Lab-Tek II chambered coverglass or at 100,000 cells per well in a 24 well plate. Cells were incubated at 37°C for 48 hr. Cells were washed 1x in PBS followed by treatment with 10 mM methyl-ß cyclodextrin (Sigma), 1 mM water-soluble cholesterol (Sigma), or a combination of 10 mM methyl-ß cyclodextrin and 1 mM water-soluble cholesterol in serum free DMEM media. Cells were treated for 1 hr at 33°C followed by three washes with PBS. Cells plated on chambered coverglass were infected with azido-DIM labeled M. marinum at an MOI of 5 for 24 hr at 33°C followed by imaging on a Nikon A1R confocal microscope with a 20x Plan Apo 0.75 NA objective. 2 µm z-stacks were generated through the infected cells. Azido-DIM spreading was calculated similar to above (Section: Confocal microscopy and image-based quantification of infection). Cells plated on 24-well plates were rescued in DMEM + 10% FBS for 3 hr at 33°C. Cells were then harvested for quantification of cholesterol levels.
3
Cell Adhesion and Spreading on Synthetic Bone Matrix
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Osteo Assay surface (synthetic bone matrix) 24-well microplates (Corning) were used. For adhesion, 24 hour-old preconditioned media from corresponding experimental groups were collected, and then 200 μL was pre-absorbed to the Osteo plates for 1 hour prior to adding 1.0 × 106 cells in 100 μL fresh media, and incubated for 30 min. Plates were gently tapped to remove loosely bound cells, and the remaining cells were stained with DAPI and enumerated by microscopy with large-stitch imaging using a 10X Plan Apo 0.75 N objective (Nikon) and an ORCA-Flash 4.0 V2 cMOS camera (Hamamatsu). For spreading measurements on synthetic bone matrix, the above protocol was used except imaging commenced immediately upon addition of cells to the plate. Cell area was quantified at 6 min intervals by manual tracing in Elements software (Nikon), using a 20X Plan Apo 0.75 NA objective (Nikon) and a CoolSNAP MYO CCD camera (Photometrics).
4
Cell Adhesion and Spreading on Synthetic Bone Matrix
5
In vivo Zebrafish Live Imaging
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Zebrafish were anesthetized in fish water containing tricaine and then and mounted onto optical bottom plates (MatTek Corporation, P06G-1.5-20-F) in 1% low melting point agarose (Invitrogen, 16520-100) as previously described (Takaki et al., 2013) . Microscopy was performed using a Nikon A1 confocal laser scanning confocal microscopy with a 20x Plan Apo 0.75 NA objective and a Galvano scanner, acquiring 30-80 μm z-stacks with 2-3 μm z-step intervals. Timelapse microscopy was performed at physiological temperature using a heat chamber set to 28°C (Okolab) with an acquisition interval of 3 minutes.
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